Deleterious Effects Of Urea Research Articles

Microscopic significance of hydrophobic residues in the protein-stabilizing effect of trimethylamine N-oxide (TMAO).

Although it is widely known that trimethylamine N-oxide (TMAO) stabilizes the native structure of proteins, the underlying mechanism of its action is poorly documented. To obtain an in-depth understanding of this important osmolyte molecule, we conducted large-scale molecular dynamic simulations of model proteins, namely, wild-type villin headpiece protein HP35 and its doubly norleucine-substituent mutant (Lys24/29Nle) HP35NN in pure urea and urea + TMAO mixed solutions for direct comparison. From extensive sampling, the protective capability of TMAO was well captured in the simulations, where HP35NN demonstrated a significantly more stable native structure than HP35 in the presence of TMAO, whereas in pure urea solution, the former denatured in a shorter time. These findings highlight the importance of the two norleucine residues that regulates the interactions of proteins with urea and TMAO. By accessing the hydration and conformational dynamics of both proteins, we were able to directly probe how TMAO compensates the denaturing effect of urea at the atomic level. The accumulation of urea around hydrophobic residues is clearly suppressed, which indicates that the van der Waals interactions between urea and proteins are weakened by TMAO. As a consequence, the hydrophobic core of protein is preferentially protected by TMAO against urea attack. Although the hydrogen bonds (H-bonds) between proteins and urea are suppressed by TMAO, this plays a very minor role than expected in the enhanced protein stability. In addition, TMAO was found to be always excluded from the protein surface and incapable of forming H-bonds with proteins. Thus, the present study provides solid evidence to support the indirect mechanism of TMAO counteracting the denaturing effects of urea.

Interactions of S-peptide analogue in aqueous urea and trimethylamine-N-oxide solutions: A molecular dynamics simulation study

The ability of the osmolyte, trimethylamine-N-oxide (TMAO), to protect proteins from deleterious effect of urea, another commonly available osmolyte, is well-established. However, the molecular mechanism of this counteraction is not understood yet. To provide a molecular level understanding of how TMAO protects proteins in highly concentrated urea solution, we report here molecular dynamics simulation results of a 15-residue model peptide in two different conformations: helix and extended. For both conformations, simulations are carried out in pure water as well as in binary and ternary aqueous solutions of urea and TMAO. Analysis of solvation characteristics reveals direct interactions of urea and TMAO with peptide residues. However, the number of TMAO molecules that enter in the first solvation shell of the peptide is significantly lower than that of urea, and, unlike water and urea, TMAO shows its inability to form hydrogen bond with backbone oxygen and negatively charged sidechains. Preferential accumulation of urea near the peptide surface and preferential exclusion of TMAO from the peptide surface are observed. Inclusion of osmolytes in the peptide solvation shell leads to dehydration of the peptide in binary and ternary solutions of urea and TMAO. Solvation of peptide residues are investigated more closely by calculating the number of hydrogen bonds between the peptide and solution species. It is found that number of hydrogen bonds formed by the peptide with solution species increases in binary urea solution (relative to pure water) and this relative enhancement in hydrogen bond number reduces upon addition of TMAO. Our simulation results also suggest that, in the ternary solution, the peptide solvation layer is better mixed in terms of water and urea as compared to binary urea solution. Implications of the results for counteraction mechanism of TMAO are discussed.

Effects of diet type on establishment of pregnancy and embryo development in beef heifers

The objectives were to determine the effects of elevated blood urea concentrations on: (i) the response to superovulation, fertilisation rate, and early embryonic development in beef heifers, and (ii) embryo survival from days 7 to 35 of gestation. In Experiment 1, heifers (18–24 months) were allocated at random (n=20 per treatment) to one of the following diets: (i) ad libitum grass silage plus 5kg commercial beef concentrates per day (controls); (ii) ad libitum grass silage plus 5kg concentrates and 250g feed grade urea per day (HE/HU); or (iii) ad libitum wheaten straw plus 250g feed grade urea and 50g vitamin/mineral mix per day (LE/HU). Serum urea concentrations were monitored throughout the experiment. Oestrus in heifers was synchronised using an intravaginal releasing device (CIDR®, InterAg, New Zealand). Oestrus was detected and in vitro produced blastocysts (day 7, morphological grades 1 and 2) were transferred to the heifers 7 days later (19 days after start of treatment diets). The heifers were maintained on the dietary treatments for a further 28 days, when pregnancy status was determined by transrectal ultrasonography. Detected pregnancies were terminated using 15mg luprostiol and recycled for Experiment 2. In Experiment 2, following a 14-day dietary rest period, the heifers were re-allocated at random to the three dietary treatments above. Heifers were treated with a CIDR for 8 days and 15mg luprostiol was given 12h before pessary withdrawal. They received 144mg pFSH (Folltropin®-V, Vetrepharm, Canada) given as 8 injections over 4 days commencing on day 6 of CIDR/dietary treatment. Heifers were artificially inseminated 48h after progesterone pessary withdrawal using commercial semen of proven fertility by a competent inseminator. The heifers were maintained on their diets until slaughter, 3 days post insemination when corpora lutea numbers were determined and embryos were recovered and cell numbers determined visually. Serum urea concentrations were greater in heifers on LE/HU than in those on HE/HU diets, which in turn were greater than controls (7.1±0.5, 4.9±0.3 and 3.2±0.1mmol/L, respectively; P<0.05). There was no effect of diet type on pregnancy rate at day 35 (42%, 47% and 46%) and on the number of corpora lutea following superovulation (5.2±0.8, 5.8±1.5 and 6.8±1.1) for heifers on control, HE/HU and LE/HU diets, respectively. The total number of embryos recovered per heifer was not different between the three groups (2.7±0.6, 3.4±1.1 and 4.8±0.8 for heifers on control, HE/HU and LE/HU diets, respectively; P>0.05), but the number of embryos with 8 or more cells at recovery was greater in heifers on LE/HU than on control diets (3.4±0.8 compared with 1.0±0.3; P<0.05). However the percentage of embryos recovered with 8 or more cells was not different between groups (70.0±13.3, 86.9±7.2 and 76.5±7.9%, for heifers on control, HE/HU and LE/HU diets respectively). Fertilisation rate, expressed as the proportion of embryos with more than one cell at recovery relative to the total number of embryos recovered, was less in the heifers on the control diet than in the other two dietary treatments (61.3±11.8, 92.0±3.5 and 86.8±5.4% for heifers on control, HE/HU and LE/HU diets, respectively; P<0.05). Deleterious effects of urea on reproduction were not found, suggesting that adverse effects of urea are likely to take place at the early oocyte development stage prior to ovulation or fertilisation following an increase in protein intake.

Refolding of urea-induced denaturation of model proteins by trimethylamine N-oxide

The biomolecules are known to be stabilized by osmolytes, trimethylamine-N-oxide (TMAO) while urea, destabilizes the protein structures. The deleterious effect of urea on proteins has been counteracted by TMAO is well understood; nonetheless, refolding of urea-induced conformational changes of proteins by TMAO is still an active subject. To understand the refolding ability of TMAO from urea-induced denaturation of biomolecules, we have performed transfer free energy (Δ G′ tr) and the hydrodynamic diameter ( d H ) of cyclic dipeptides (CDs) such as, cyclo(Gly–Gly), and cyclo(Leu–Ala) through solubilities and dynamic light scattering (DLS) measurements, respectively. We observed positive and negative values of Δ G′ tr for CDs from water to TMAO and urea, respectively. Our results reveal that TMAO is a refolding additive for urea deleterious actions on CDs at 1:1 and 1:2 molar ratios of TMAO and urea. However, TMAO (1 M) fails to refolding CDs structure from the urea (3–5 M)-induced conformational changes on CDs.

Trehalose protects urea-induced unfolding of α-chymotrypsin

Trehalose, a naturally occurring osmolyte, appears to be one of the most effective protectants for enzymes under various stress conditions while urea, a classical denaturant, destabilizes the activity, function, and alters the native structure of proteins. Herein, we have characterized the counteracting effects of trehalose on the deleterious effect of urea on α-chymotrypsin (CT) through the calorimetric data (transition temperature ( T m), enthalpy change (Δ H), heat capacity change (Δ C p) and Gibbs free energy of unfolding (Δ G u) by using differential scanning calorimeter (DSC) and circular dichroism (CD) techniques, respectively, at a 1:2 ratio of trehalose and urea, as well as various urea concentration (up to 6 M) in the presence of 1 M trehalose. Our parallel experimental results explicitly elucidate that trehalose strongly offset the deleterious actions of urea on CT at 1:2 molar ratio of trehalose and urea, however, trehalose (1 M) some how failed to counteract the perturbation effects of urea (3–6 M) on CT.

Osmolyte Counteracts Urea-Induced Denaturation of α-Chymotrypsin

The stability of proteins is reduced by urea, which is methylamine and nonprotecting osmolyte; eventually urea destabilizes the activity and function and alters the structure of proteins, whereas the stability of proteins is raised by the osmolytes, which are not interfering with the functional activity of proteins. The deleterious effect of urea on proteins has been counteracted by methylamines (osmolytes), such as trimethylamine N-oxide (TMAO), betaine, and sarcosine. To distinctly enunciate the comparison of the counteracting effects between these methylamines on urea-induced denaturation of alpha-chymotrypsin (CT), we measured the hydrodynamic diameter (d(H)) and the thermodynamic properties (T(m), DeltaH, DeltaG(U), and DeltaC(p)) with dynamic light scattering (DLS) and differential scanning calorimeter (DSC), respectively. The present investigation compares the compatibility and counteracting hypothesis by determining the effects of methylamines and urea, as individual components and in combination at a concentration ratio of 1:2 (methylamine:urea) as well as various urea concentrations (0.5-5 M) in the presence of 1 M methylamine. The experimental results revealed that the naturally occurring osmolytes TMAO, betaine, and sarcosine strongly counteracted the urea actions on alpha-chymotrypsin. The results also indicated that TMAO counteracting the urea effects on CT was much stronger than betaine or sarcosine.

Effect of diet quantity and urea supplementation on oocyte and embryo quality in sheep

The objective was to investigate the effects of dietary energy and urea supplementation on oocyte and embryo quality in sheep using in vivo and in vitro experimental models. Sixty-three ewes were fed grass metal at 0.5 or 2.0 times maintenance energy requirements (MER). The diet was supplemented with feed grade urea (U) for half of the ewes on each energy treatment. Ewes were stimulated with 1000 IU eCG and either slaughtered on the day of pessary withdrawal for in vitro embryo production, or mated and slaughtered on Day 5 for embryo recovery. Urea decreased cleavage rate (48.3 vs 39.7%) and consequently blastocyst rate (41.6 vs 36.8%) but the differences were not significant. Oocytes from animals on 2.0 MER had a lower cleavage rate (54.9 vs 36.0%) and blastocyst yield (49.3 vs 31.4%) than those on 0.5 MER. However, there was an interaction between urea and energy for cleavage (P = 0.04) and blastocyst yield (P = 0.03) indicating a variable response to urea in the presence of high energy. This was manifested by a decrease in cleavage rate in the presence of urea and high energy (22%, 8 of 36), and a reduction in blastocyst development (19%, 7 of 36). When blastocyst development rate was expressed as a proportion of cleaved oocytes there was no difference between groups; in addition, there was no difference between groups in terms of blastocyst hatching rate (overall mean 66.1%) or blastocyst cell number on Day 8 (overall mean ± SEM, 138.4 ± 9.0, n=61). The effect of urea on cleavage rate in vivo was more severe. Urea supplementation reduced (P<0.001) the cleavage rate (93 vs 62%). Despite this, the yield of blastocysts was unaffected. Oocytes from ewes on 0.5 MER exhibited a lower (P<0.05) cleavage rate than those on 2.0 MER (66 vs 87%). This effect was also apparent at the blastocyst stage (40.0 vs 50.9%), although the difference was no longer significant. There were no differences in hatching rate (overall mean 70.7%) or blastocyst cell numbers (overall mean ± SEM, 166.3 ± 15.6 n=40). Collectively, these results suggest that both high dietary energy and urea content influence subsequent embryo development in vitro, and the deleterious effects of urea are likely influenced by concomitant energy intake.

Import into mitochondria of precursors containing hydrophobic passenger proteins: pretreatment of precursors with urea inhibits import

We have studied the import into isolated yeast mitochondria of three hydrophobic passenger proteins attached to the N-terminal cleavable presequence of mitochondrial ATPase subunit 9 from Neurospora crassa. One natural precursor (pN9) contained N. crassa subunit 9; two chimaeric precursors, N9L/Y8-1 and N9L/Y9-2, respectively contained yeast mitochondrial ATPase subunits 8 and 9. In the absence of urea, pN9 and N9L/Y8-1 are imported efficiently but N9L/Y9-2 is not imported. After pretreatment of precursors in 4 M urea, binding of pN9 to mitochondria is marginally affected while its import is substantially inhibited; the binding to mitochondria of chimaeric proteins, N9L/Y8-1 and N9L/Y9-2, is greatly enhanced but no import is observed. This behaviour of import precursors containing hydrophobic passenger proteins is contrasted with that of a hydrophilic chimaeric precursor pCOXIV-DHFR, whose binding and import are enhanced by pretreatment with a high concentration of urea (8 M). The import of N9L/Y8-1 is very sensitive to the presence of low concentrations of urea in the import reaction mixture, and is abolished above 0.5 M urea although precursor binding to mitochondria is increased. By contrast, neither the import nor binding of pCOXIV-DHFR is affected directly by urea up to 0.8 M. These deleterious effects of urea on import of the chimaeric precursors N9L/Y8-1 and N9L/Y9-2 are interpreted in terms of a non-productive binding of these precursors to mitochondria, brought about by exposure of their hydrophobic domains resulting from urea unfolding. The generalization that membrane translocation of mitochondrial import precursors is enhanced by their prior unfolding in urea thus does not apply in the case of these precursors containing hydrophobic passenger proteins.

Deleterious effects of urea on glycolytic rates and ventricular function in isolated perfused rat heart

Deleterious effects of urea on glycolytic rates and ventricular function in isolated perfused rat heart

Effects of urea on microflora and on cleistocarps of Sphaerotheca mors-uvae on detached black-currant leaves

There was a rapid degeneration of asci and ascospores of Sphaerotheca mors-uvae (Schw.) Berk, when black-currant leaf disks bearing cleistocarps were dipped in 5% urea and then kept at various temperatures in the laboratory. No such degeneration occurred when leaf disks, similarly treated in autumn 1968, were set out in the field in plastic-net bags, or when disks, cut from naturally overwintered leaves, were treated and placed in the field in the spring of 1969. There were temporary increases in the bacterial populations of the treated disks in both instances. In similar field experiments in autumn 1969 and spring 1970, treatment of leaves with urea resulted in a rapid degeneration of asci and ascospores within cleistocarps and also in marked increases in the total numbers of bacteria and in the numbers of chitinoclastic bacteria and actinomycetes. The deleterious effect of urea probably results mainly from a direct effect on the ascus membranes, though the stimulation of the microflora, especially chitinoclastic micro-organisms, may contribute in part. Treatment of fallen black-currant leaves with urea is unlikely to be a practical means of limiting the perennation of S. mors-uvae because the urea is rapidly leached by rain. Such leaching probably accounted for the lack of effects in the 1968–69 experiments.